The present invention relates to imaging the sequencing of DNA fragments. In particular, the present invention is directed to a DNA sequencing technique using a full-width array scanner or large area detector to selectively image an entire gel sequencing process.
In general, modem high-speed DNA sequencers facilitate the migration of DNA fragments attached with fluorescent labels along a gel to achieve separation therebetween. Useful chemical base sequencing information is obtained by the sequences by detecting fluorescence of the fragments at a fixed location. Methods have been developed to most efficiently distinguish the four bases (adenine, thiamine, guanine and cytosine) of interest in DNA sequencing. DNA fragments are identified using a fluorescent dye label for each of the four chemical bases.
Generally, a method known as electrophoresis is used for the above-referenced separation of DNA fragments. Electrophoresis is a separation technique accomplished by inducing the migration of charged molecules (or particles) in an electrolyte under the influence of an electric field. Smaller or more highly charged sample molecules move faster than larger or lower charged molecules. By utilizing this technique, each species of a DNA sample molecule is divided into bands that pass or reach a fixed point at different times. Once these bands pass the fixed point, they are analyzed using a detection method in order to sequence the DNA fragment.
Different types of electrophoresis are used in DNA sequencing. For example, in capillary electrophoresis, a buffer-filled capillary is suspended between two reservoirs of buffer. An electric field is applied to both ends of the capillary to create a bias across the length of the capillary. A sample is introduced at one end of the capillary, typically the capillary end with a higher electric field potential. The sample migrates according to the physical characteristics of the DNA fragments and separate along the electric field bias created.
As a further example, the standard high speed automated DNA sequencers (such as the Applied Biosystems Model 377) make use of a thin gel layer, of several hundred microns, encased between two etched glass plates in order to separate the fluorescent dye labeled DNA fragments by an applied electric field. The separation of the DNA fragments depends on their relative lengths, which can vary by as little as one chemical base. Making use of enzyme chemistry and labeling each of the four bases, by terminating a given DNA fragment with a differently colored dye, the process is accomplished.
Further, in another exemplary technique known as gel electrophoresis, an electrolyte is usually supported by a hydrophilic matrix, the gel, which is coated on a sheet of glass then placed in contact (sandwiched) with another glass plate and finally sealed on each side of the glass plates with a gasket. The samples containing the DNA fragments are applied to the top edge of the gel. The bottom edge of the gel plates is placed vertically in a reservoir containing a buffered electrolyte. A second reservoir is then placed on the top of the glass plates and also filled with a buffered electrolyte. A current is then run to each reservoir by an electrode connected to a power supply. Typically, a gel run will take close to six hours. After the run, the plate is stained in order to visualize the bands of interest.
As is understood, using any of these electrophoresis methods, each fluorescent dye label can be individually detected by a detector. Once a DNA fragment migrates, it is detected by the detector and then the identity of the base is determined based on the fluorescent dye label.
One method that is known to accomplish this determination is a one-color, four-intensity scheme. This scheme is not desirable, however, in controlling the polymerase and in maximizing the signal-to-noise ratio. A two-color, two-intensity method has also been developed which has advantages over the one-color scheme. These advantages include simpler optical arrangement, good light collection and a straightforward algorithm. However, the two-color method still has disadvantages in controlling the polymerase. Thus, the most commonly used technology is a four-color method that uses four standard dyes for each of the chemical bases of DNA.
In addition, as expressed above, to effectively sequence the DNA fragments that are placed within the separation apparatus, a suitable detection system is necessary. Conventionally, a detector is positioned at the end of the migration lanes. The detector then detects each DNA fragment as it migrates to the end, thereby sequencing the DNA fragments. A common disadvantage of all known detection techniques, however, is that they consume an excessive amount of time.
For example, in the case of detection of a capillary electrophoresis system, the light is collected from the end of the capillary tube where the DNA fragments migrate. Detection is accomplished by putting a lens in front of the fixed point and a detector behind the lens. The detector collects all of the light that was emitted through the lens. However, in this system, not all of the light is collected and detected, which poses a particular problem if the size of the emission is large. Moreover, the time required to accurately detect in this fixed end detector is excessive.
Another older technology used the separation of radioactive isotope labeled DNA fragments followed by overnight exposure to special film. This technology showed that over 300 bases were present in the entire gel after high field electrophoresis lasting seventeen minutes. However, this process of detection is also excessively time consuming.
Other devices use a single photomultiplier tube (PMT) imager scanned across the plate, or a charge-coupled device (CCD) to image a line. In both situations, the time necessary for imaging is excessive. In the case of a CCD, the detector is optically coupled to the capillary array by way of the capillaries in the array being optically coupled to the linearly aligned pixels. A sample containing a fluorescent target species, such as a DNA fragment, is introduced into the intake end of the optically coupled capillary such that it migrates through the capillary toward the outflow end. Fluorescence emission from the target species is then induced by irradiating it with a beam of coherent light. Fluorescence emission is detected by the image array detector through the transparent portion of the optically coupled capillary using the optically coupled pixels.
The detection systems of the prior techniques pose many problems in the efficiency of DNA sequencing and imaging. The prior systems are very slow. For example, some fixed end detection systems require up to eight hours in order to detect one sample. Further, by using a prior art detector, all of the possible data is not collected. For example, the relationship between the DNA fragments cannot be recorded as they migrate from one end of the separation apparatus to the other end. Another reason that the prior art detection systems are not efficient is that these systems typically only detect one band at a time, e.g. the band that has reached the end of the separation apparatus in fixed end detection. This provides no information about where other bands have migrated with respect to the bands that are being detected.
The present invention overcomes the above-referenced problems and others and provides an improved method for the imaging of DNA fragments for DNA sequencing.
The present invention provides a technique for sequencing an entire sequencing plate or separation apparatus holding DNA fragments using known methods of DNA sequencing in combination with a scanner or large area detector. The resultant system and method improve speed of detection and processing and more accurately achieve sequencing of DNA fragments.
In one aspect of the invention, the method comprises steps of placing a DNA sample within a buffer in separation apparatus, applying an electric field across the separation apparatus to create a bias in the buffer such that the DNA sample migrates from one end of the apparatus to another end along a migration channel, separating the DNA sample into fragments along the migration channel within the buffer, detecting fluorescent light emitted from the fragments along the migration channel and generating a full image of the separation apparatus and the separated DNA fragments based on the detecting.
In another aspect of the invention, the apparatus comprises a separation apparatus operative to receive a DNA sample and facilitate migration and separation into fragments of the DNA sample along a migration channel within the apparatus, a detector operative to detect light emitted the DNA fragments along the migration channels and an image processor operative to generate image data representing a full image of the separation apparatus and the fragments.
In another aspect of the invention, the detector is a full width linear scanner.
In another aspect of the invention, the detector is an amorphous silicon array.
One advantage of the present invention is to speed up DNA sequencing by fluorescence labeling using scanning and/or large area detection.
Another advantage of the present invention is to use multiple scans of the capillary electrophoresis gel to calculate an optimum combination of electrophoresis and two-dimensional imaging steps to speed up the sequencing process.
Yet another advantage of the present invention is that many different plates may be monitored at the same time, as the scanner may scan an entire plate in a short time.
Yet another advantage of the present invention is that the optical efficiency of the lens system employed is much more efficient by having greater light collection.